We will address the challenge, "Enabling Technologies (06)", and the specific topic, "Small RNAs (06-GM- 105)", described by "the identification and functional characterization of all classes of small RNAs, to elucidate their regulation and mechanism of action and to understand their evolutionary origin." Riboswitches have the remarkable ability to detect a variety of fundamental metabolites and control the expression of their own protein, based on the conditions in their environment. Because riboswitches are highly specific, they can easily discriminate between similar small molecules. A riboswitch has the ability to produce concentration-dependent promotion or suppression of genes, giving it more precise control over gene expression than, for example, RNA interference (RNAi), which may causes permanent gene silencing. A gap in knowledge exists between known biological function and mechanistic understanding of riboswitches. While it is known that the riboswitch must switch, it is not known how the riboswitch is able to switch. Little is known about how the metabolite is able to control the global fold of the aptamer domain. No detailed information concerning fundamental questions of ligand effects on folding rate, domain conformation or mechanism of stabilization have been published. Computationally, the field is almost unexplored. We have published the first and only all- atom simulations of a riboswitch to date. While a few limited studies of binding have been performed on the isolated sensor domain of purine riboswitches, no mechanistic experimental studies of switching have been performed on a full riboswitch. We will use an approach that integrates experiment and simulation to uncover the principles of operation for the SAM riboswitch. Using a novel all-atom reduced-potential simulation technique and a novel fluorescence-based switching assay, we will investigate the interplay between folding, ligand-binding and conformational switching. Experimental data from biochemical experiments will be incorporated into force field potentials. Iteration between experiment and simulation will generate simulations consistent with experiments to provide a structural dynamics interpretations of experimental data and to make testable predictions. As the design of synthetic riboswitches has proceeded by trial-and-error, our study will provide principles for designing new riboswitches. These synthetic switches would have far reaching applications from synthetic biology to drug delivery and therapeutic. Coarse-grained and large-scale all-atom simulations based on basic physics principles will be used to design and interpret rapid kinetics experiments. The high performance computing capability of LANL will be leveraged to gain unique insight into the switching process. Our results on the molecular mechanism will shed light on the more general problem of gene regulation by non-coding RNAs. With respect to therapeutic design, at least 16 human bacterial pathogens rely on riboswitch-controlled gene expression. Focusing on the rapidly growing area of non-coding RNA, our project will recruit new talent and stimulate the economy by creating 2 new jobs and by purchasing a $60,000 stopped- flow instrument from the American company, Kintek. PUBLIC HEALTH RELEVANCE: Riboswitches are recently discovered powerful noncoding RNAs that turn on or off gene expression, depending on the presence of a ligand. We will use an approach that integrates experiment and simulation to uncover the detailed mechanism of switching. In particular, we will explore the competition between ligand-binding, folding, and alternative secondary structure formation. The action mechanism of the SAM riboswitch will be explored by integrating biochemical experiments with large-scale computer simulations. The interplay between folding, ligand-binding and conformational switching will be studied with a combination of biochemical protection, rapid kinetics and large-scale molecular simulation.